High Electrochemiluminescence from Ru(bpy)3 2+ Embedded Metal–Organic Frameworks to Visualize Single Molecule Movement at the Cellular Membrane

Abstract Direct imaging of single‐molecule and its movement is of fundamental importance in biology, but challenging. Herein, aided by the nanoconfinement effect and resultant high reaction activity within metal–organic frameworks (MOFs), the designed Ru(bpy)3 2+ embedded MOF complex (RuMOFs) exhibits bright electrochemiluminescence (ECL) emission permitting high‐quality imaging of ECL events at single molecule level. By labeling individual proteins of living cells with single RuMOFs, the distribution of membrane tyrosine‐protein‐kinase‐like7 (PTK7) proteins at low‐expressing cells is imaged via ECL. More importantly, the efficient capture of ECL photons generated inside the MOFs results in a stable ECL emission up to 1 h, allowing the in operando visualization of protein movements at the cellular membrane. As compared with the fluorescence observation, near‐zero ECL background surrounding the target protein with the ECL emitter gives a better contrast for the dynamic imaging of discrete protein movement. This achievement of single molecule ECL dynamic imaging using RuMOFs will provide a more effective nanoemitter to observe the distribution and motion of individual proteins at living cells.

. Setup of the electrochemical (ECL) chip. Figure S2. DLS verification of the synthesized RuMOFs. Figure S3. TEM energy mapping of ZrMOF NPs and RuMOF nanoemitters. Figure S4. Zeta potential of the ZrMOF NPs and RuMOFs. Figure S5. Photographs of ZrMOF NPs and RuMOF nanoemitters in water. Figure S6. Stability verification of the ZrMOF NPs and RuMOFs. Figure S7. The peak intensity analysis of ECL signal spots from marked region. Figure S8. ECL intensity distribution of ECL nanoemitters. At least 600 ECL nanoemitters were analyzed. Figure S9. Measure of the Number of Ru(bpy)3 2+ molecules per RuMOF. Figure S10. ECL images of ECL nanoemitters and the representative ECL-time trajectory obtained from single ECL nanoemitter. Figure S11. Geometry modeling of RuMOF in COMSOL software. Figure S12. Simulated concentration distribution of Ru(bpy)3 2+ * and 3D distribution pattern of ECL emission. Figure S13. The verification of the accuracy and effectiveness of ECL nanoemitters approach monitoring single proteins.. Figure S14. Characterization of the bio-application performance of ECL nanoemitters on living cells. Figure S15. Characterization of the optical performance of ECL nanoemitters on living cells. Figure S16. Validation of single protein signals on the surface of living cells. Figure S17. Characterization of the imaging performance of ECL nanoemitters on living cells.. Figure S18. Statistical analysis of proteins in different regions on single cell and the temporal evolution record of protein individuals, Figure S19. Dynamic analysis of protein individuals in different regions of HeLa cells. Table S1. Reactions involving the ECL process.

Apparatus and characterization
An Agilent HP8453 UV-vis spectrophotometer was used to record UV-vis absorption spectra. The fluorescence measurements were performed using a quartz cuvette and an F-4600 fluorescence spectrometer (Japan). The shape and size of Uio-67 were characterized with a JEOL JEM-2011 transmission electron microscope (TEM). A Zetasizer (Nano-Z, Malvern, UK) was used as Zeta potential and grading analysis. The single molecule and ECL imaging experiments were achieved with a home-built imaging system by Professor JW Liu.

Synthesis of nanoconfined-RuMOFs nanoemitters
The method of synthesising RuMOFs has been improved refer to previous works. 1,2 As the precursor solution of organic ligand, 45 mg of BPDC was dispersed into 20 mL of DMF contained 360 μL of triethylamine. As the white metal precursor solution, 45 mg of ZrCl4 was dispersed into 16 mL of DMF with ultrasonic. Then, the two solutions were mixed with 2.25 mL of acetic acid, and the mixture was transferred into a Teflon sealed autoclave to obtain a high-pressure reaction and sat at 90 °C for 24 h. After the solvothermal reaction, the products were centrifuged at 6500 rpm for 12 min, then further washed with DMF, methanol and ethanol respectively. Ultimately, the white precipitate was obtained. The product was dried and used for further experiments or dispersed at ethanol with a concentration of 2 mg/mL. Whereafter, the tris-(2,2′-bipyridine) ruthenium (II) hexafluorophosphate (Ru(bpy)3(PF6)2) was incorporated into the ZrMOF nanoparticles. Simply, 10 mL of Ru(bpy)3(PF6)2 DMF solutions (2 mg/mL) was mixed with 10 mL of the UiO-67 dispersion to obtain orange yellow solution. The solution was then stirred at 95 °C for 15 h, followed by removal of the DMF solvent and centrifugation with DMF and ethanol. The obtained yellow solid was dried or dispersed in ethanol and store at 4 °C for further work.

Preparation of nanoconfined-ECL nanoemitters
The RuMOFs were first washed with ultrapure water twice (6500 rpm, 12 min) to remove ethanol and resuspended in DMF. The phosphate-terminal labeled aptamers (phosphate-T10-Sgc8 and phosphate-control) (100 µL, 10 µM) were added to freshly synthesized RuMOFs (2 mg/mL) and the mixture was incubated under gentle shaking for 5 h at room temperature. Then, free aptamers were removed by centrifugation and washing with water, the conjugates were redispersed in buffer/water. Finally, the ECL probe was obtained and stored at 4 °C until use.

Preparation of Aminated ITO
Firstly, the indium tin oxide (ITO) coated cover slips (0.13-0.17 mm thick, 20 × 20 mm, ~ 15-30 Ω, SPI Supplies, USA) were cleaned by sonication in acetone, ethanol, and ultra-pure water for 12 min respectively. Then, they were dried by N2 stream and etched in vacuum plasma (PDC-002, Harrick Plasma Inc., U.S.) for 8 min. Whereafter, the ITO slides were dipped into the mixture solution of H2O2/NH3·H2O/H2O (1:1:5, V/V) for 0.5 h to generate an active hydroxyl layer and washed twice times with ultrapure water. After drying by a N2 stream, the ITO slides were immersed in 0.5% APTES (ethanol) for 0.5 h at room temperature, ultrasonized 30 s and rinsed twice quickly with ethanol, and heated at 80 °C for 30 min. Finally, after the washing with ultrapure water, the amine groups were successfully introduced on the ITO electrode.

Fabrication of the ECL detection chip
The simple and convenient detection chip was composed of an ITO electrode, a square PDMS and a carbon strip. Firstly, four holes were punched at the central part of a square PDMS by puncher as reservoirs. Then, the PDMS was etched in vacuum plasma for 10 min. Afterwards, the PDMS layer was attached to the modified ITO piece surface and pressed firmly. Then, with conductive silver paste (SPI, USA), a carbon strip was attached to one corner of the modified ITO. The prepared chips were stored for further use.

Construction process of ECL biosensor
A sandwich interface was constructed on the aminated ITO slice by stepwise modification. 30 μL of 5% GLD was added into the detection chip to activate the amine groups for 1 h at room temperature. Then, 30 μL of 0.1 μg/mL Ab1 was introduced onto the GLD/ITO activated surface by covalent coupling. After washing gently with 0.01 M PBS, the detection chip was exposed to 30 μL of 1% BSA for 1h at 37 °C. Whereafter, different concentrations of PTK7 solution (30 μL) were added to this detection chip and left at 37 °C for 1h. 30 μL of ECL emitters were then dropped onto this detection chip and incubated at 37 °C for 0.5 h. Finally, the chips were washed thoroughly with PBS to remove free ECL probe or nonspecifically bounded conjugates.
Cell culture and seeding CCRF-CEM cells, Ramos cells and Hela cells were cultured in RPMI 1640 medium, with 10% fetal bovine serum and 0.5 mg/mL penicillin-streptomycin at 37 °C under a 5% CO2 atmosphere. For the suspension cells (CEM cells and Romas cells), the ITO electrodes were etched in vacuum plasma for 6 min and then immersed into 0.01% (w/v) PLL solution for a 10 min incubation. This step could can increase cell adhesion of the sensing interface. 3 The CEM cells and Romas cells in the exponential growth phase were centrifuged at 1000 rpm for 3 min and washed with PBS. The cell number was determined using a hemocytometer. Then, cells were incubated in cell culture medium containing the ECL probes (25 µg/mL), which were shaked gently for 45 min h at 37 °C to label the probes on cell surface through the specific recognition of Sgc8 aptamers and PTK7 protein on cell surface. The medium was then removed and the cells were washed gently with PBS. After that, the suspension cells were seeded on the pole of the PLL/ITO and incubated 0.5 h for ECL imaging. For the adherent cells (Hela cells), the ITO electrodes were etched in vacuum plasma for 6 min. The Hela cells in the exponential growth phase were digested for 2 min by trypsin (0.1%, m/v), and then separated from the cell culture medium. The cells were gathered by the centrifuging at 800 rpm for 2 min, and then, washed with PBS. The cells were then resuspended in cell culture medium and seeded on the pole of the ITO. 10 h (37 °C, 5% CO2) incubation was applied for 10 h to achieve the immobilization of cells. Then, the freshly culture solution containing the ECL probe (25 µg/mL) was added and incubated for 60 min at 37 °C. After washing cells, the single cell ECL imaging were performed.

Measure of the number of Ru(bpy)3 2+ per RuMOFs
First, we collected the photoluminescence information of a single Ru(bpy)3 2+ molecule, as shown in Figure S9. Then, 500 imaging signal points were selected for PL intensity analysis to obtain the average intensity ( 0.85) of a single Ru(bpy)3 2+ (Figure S9). Under the same imaging and analysis conditions, the PL signals of 500 emitters were statistically analyzed to obtain their intensity information ( 510 31), as shown in Figure S8. After comparing the intensity with that of a single Ru molecule, the amount of Ru molecules contained in each RuMOF was obtained ( 600 ± 37). W represents for half-peak width intensity value.

Imaging instrumentation and data analysis
The PL/ECL/optical imaging was operated on a homemade imaging system, which was composed of a wide-field epi-fluorescence microscope (Chiyoda, Tokyo, Japan), an ultrasensitive electron multiplying charge coupled device (EM-CCD) (Ixon DU888, Andor Technology Plc., U.K.) and a ECL detection chip, shown in Figure S1. The microscope was installed in a self-made dark box to avoid interference from outside light. A motorized microscope stage and oil immersion objective len (NA 1.45, 100×, Olympus, Japan) was equipped for the sample recording. ECL detection chip was used in a two-electrode configuration, in which the modified ITO electrode acted as working electrode, and a Pt wire as counter electrode. Moreover, this imaging system included a potentiostat from the voltage generator (DG 1021, Rigol, China). A switching of the potential between +1.35 V and 0 V was continuously employed on the work electrode to trigger the ECL reaction. TPrA (0.08 M) was chosen as an oxidative-reduction sacrificial coreactant. The EMCCD was triggered and synchronized by the potentiostat. All measurements were performed at room temperature. The data analysis method is implemented from the Professor W.E. Moerner lab to recognize single molecule spots in images with some modification. 3,4 In this work, the data were mainly analyzed with ImageJ and home-written MATLAB.

Calculation of photon counts
The A/D (analog-to-digital) counts of the camera is described below.
where N is the number of photons (photon counts) incident to the pixel, QE is the photoelectric conversion efficiency, electronmultiplying (EM) Gain is the electron multiplication coefficient of the camera. A/D unit is the analog-to-digital conversion coefficient that is 5.67 for the EMCCD. The EM Gain selected in the experiment is 300 and the QE is viewed as 1.

COMSOL simulation for ECL pattern analysis
The experimental configuration was modeled using the commercial finite element software (COMSOL). In this simulation, a 2D axisymmetric model was built to simulate the physical geometry in ECL experiments, as shown in Figure S11. A physical geometry consisting of porous nanoparticles, electrochemical reaction cell, supporting ITO electrode and electrolyte was constructed in the electrical analysis module of COMSOL software. The boundary and domain settings were shown in Figure S11. The charge transfer reaction occurred on the surface of the supporting electrode with potential of 1.35 V (domain 2). The homogeneous chemical reaction occurred in the electrolyte (domain 1). A porous nanoparticle of diameter 100 nm loaded with Ru(bpy)3 2+ was located in the middle of the electrode. The kinetic and thermodynamic parameters were referred to previously reported works, except for the diffusion coefficient of TPrA and Ru-complex were adjusted accordingly. 5 Table S1. Reactions involving the ECL process. (

Compound
Total initial concentration Diff.coeff.